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ARTICLE IN PRESS
Biomaterials 26 (2005) 2115–2120
A novel non-toxic camptothecin formulation for
cancer chemotherapy
M. Berradaa, A. Serreqia, F Dabbarha, A. Owusub, A. Guptaa, S. Lehnertb,*
b
a
Bio Syntech Canada Inc, 475 Armand-Frappier, Laval, Que., Canada H7V 4B3
Department of Radiation Oncology, McGill University, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Que., Canada H3C 1A4
Received 15 January 2004; accepted 4 June 2004
Available online 8 September 2004
Abstract
The use of a novel injectable biocompatible and biodegradable camptothecin-polymer implant for sustained intra-tumoral release
of high concentrations of camptothecin is described. The drug delivery vehicle is an in situ thermogelling formulation, which is based
on the natural biopolymer chitosan. This formulation, containing homogeneously dispersed camptothecin, was implanted intratumorally into a sub-cutaneous mouse tumor model (RIF-l). The effectiveness of treatment was measured in terms of tumor growth
delay (TGD). Animals treated with the polymer implants containing camptothecin had signiﬁcantly longer TGDs compared to
untreated animals as well as to animals treated systemically with camptothecin by intra-peritoneal injection with no evidence of
toxicity in terms of loss of body weight. The results indicate that this novel biodegradable polymer implant is an effective vehicle for
the sustained intra-tumoral delivery of camptothecin which might also be suitable to deliver other insoluble anti-cancer drugs such
as taxol.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Drug delivery; Thermally responsive material; Biodegradation; Intra-tumoral; Chitosan; BST-gel
1. Introduction
Camptothecin is an inhibitor of the DNA-replicating
enzyme topoisomerase I [1] which is believed to act by
stabilizing a topoisomerase I-induced single strand
break in the phosphodiester backbone of DNA, thereby
preventing religation [2,3]. This leads to the production
of a double-strand DNA break during replication,
which results in cell death if not repaired. The naturally
occurring alkaloid was ﬁrst isolated from the tree
Camptotheca acuminata in China in 1966 [4]. In preclinical studies camptothecin, has been shown to be
effective against human xenografts of colon, lung,
breast, ovarian, and melanoma cancers [5–7]. But in
spite of the promise demonstrated at the pre-clinical
level, clinical trials were abandoned due to unexpected
toxicity and low antineoplastic activity [8–10]. In
addition, camptothecin was felt to have limited clinical
*Corresponding author. Tel.: +1-514-934-1934-44161; fax: +1-514934-8220.
E-mail address: [email protected] (S. Lehnert).
0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2004.06.013
potential because of its low solubility and it has been
proposed that local delivery of camptothecin would be a
means to achieve effective drug concentrations in brain
tumors without the undesirable side effects associated
with systemic delivery [11,12].
The therapeutic potential of an intra-tumoral system
for delivery of camptothecin was investigated in Fischer
rats with intra-cranially implanted 9L gliosarcoma. In
this model systemic administration of camptothecin did
not extend survival beyond that of controls, however
intra-tumoral implant of pCPP-SA wafers containing
50% camptothecin (w/w) resulted in signiﬁcantly
extended survival compared with control group
(po0:001) with 4/10 rats surviving longer than 120
days. Survival was also signiﬁcantly longer than that
seen in a group given intra-tumoral BCNU (3.8% w/w)
(po0:001). Results obtained with biodegradable polymers in this and other studies are promising, however
these devices have the disadvantage that insertion
requires surgical intervention. In some cases this can
be done when the tumor is resected during the course of
conventional treatment. Nevertheless, dependence on an
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invasive procedure remains a drawback. Another mode
of drug delivery, biodegradable microspheres avoids the
need for surgery for insertion since they can be
introduced by intra-tumoral injection [13,14]. However,
microspheres do not form a continuous ﬁlm or solid
implant with the structural integrity needed for certain
prostheses and they may be poorly retained under
certain circumstances because of their small size,
discontinuous nature and lack of adhesiveness.
During the last decade, injectable in situ gel-forming
systems have received increased interest in drug delivery
and tissue engineering. These devices can overcome
many of the problems associated with polymers or
microspheres in that they are both injectable and
produce solid biodegradable implants with a range of
mechanical characteristics in terms of rigidity and load
bearing making them compatible with both soft and
hard tissues. In the present work, we have used a
chitosan polymer to formulate a biodegradable and
biocompatible implant for controlled delivery of camptothecin in a slow-release manner directly into a mouse
ﬁbrosarcoma (RIF-l) implanted subcutaneously in C3H
mice. In this paper, we report the in vitro release
characteristics of the camptothecin-polymer implant
and the in vivo effect of delivering camptothecin in
high concentrations to a murine tumor. The delivery
vehicle used is one of a family of thermosensitive
chitosan solutions, formulated at physiological pH,
which remain liquid at low temperature and turn into
gel when heated. The polymeric matrix used in this
study consists of chitosan polymer and b-glycerophosphate. Addition of glycerol-2-phosphate (b-GP) to
chitosan solution produces a hydrogel which undergoes
sol–gel transition at a temperature close to 37 C,
making the formulation a suitable vehicle for drug
administration since the hydrogel when implanted into
the body, ﬂows to ﬁll voids or cavities and becomes solid
at body temperature. These hydrogels are suitable
carriers for water-insoluble drugs and they are nontoxic and highly biocompatible [15–17]. Chitosan is an
important natural polymer widely used for medical and
pharmaceutical applications [18].
2. Materials and methods
2.1. Materials
Chitosan (Deacetylation degree DDA. = 85% determined by 1H NMR, Mw=3 105 Da) was obtained
from shells of shrimps or lobsters as described elsewhere
[15]. Chitosan ﬂakes were dissolved in aqueous hydrochloric acid (0.1 n), ﬁltered, dialyzed and precipitated
with aqueous NaOH (6 n). The precipitated chitosan
was washed several times with water and vacuum
dried. The white chitosan powder obtained was stored
in a closed ﬂask until used. The chitosan was ultrapure with no endotoxins, proteins, inorganics and
heavy metals. Research grade camptothecin (CPT) and
b-glycerophosphate (b-GP) were obtained from Sigma
Chemicals.
2.2. Preparation of an autogelling chitosan solution :
chitosan/GP
Chitosan solutions (1.7% w/w) were prepared in 0.1 m
hydrochloric acid at room temperature. The chitosan
powders were progressively added to the solvent with
stirring and mixture was stirred for a further 3 h. Sterile
formulations were obtained by autoclaving (121 C,
20 min) [16]. To 9 ml of cooled chitosan solution, chilled
45% (w/w) b-GP aqueous solution (sterilized through a
0.20 mm ﬁlter) was carefully added dropwise to obtain
clear and homogeneous liquid solutions in a ﬁnal
volume of 10 ml. This formulation contained 1.53%
chitosan, 4.5% b-glycerophosphate. This ratio of
chitosan:GP was similar to that previously described
[17] and had a thermogelling temperature of 37 C. The
ﬁnal solutions were mixed an additional 10 min at 4 C.
The pH of the ﬁnal cold solutions ranged from 6.9 to
7.2. This clear, autogelling system is proprietary and
patented by BioSyntech [17].
2.3. Preparation of chitosan/GP loaded with
camptothecin : chitosan/GP/CPT
Homogeneous clear chitosan/hydrochloric acid solutions (1.7% w/w) were prepared, then autoclaved for
20 min at 120 C. Chitosan/CPT formulations were
prepared at room temperature by homogeneously
dispersing the powdered camptothecin in chitosan
solutions at a loading of 4.5% w/w under aseptic
conditions. Camptothecin was sterilized by g-irradiation with 25 kGy from a 60Co source (MDS Nordion
Inc. Laval, Qc, Canada). Stability of camptothecin
after gamma irradiation was conﬁrmed by HPLC
(procedure described in Section 2.7 below). Irradiated
camptothecin was stored at 4 C along with a control
sample of unirradiated powder for up to 2 months.
HPLC analysis on non-irradiated and irradiated samples gave identical results indicating that gamma
irradiation and storage had not caused any degradation
of the drug. b-glycerophosphate was dissolved in
distilled water and sterilized by ﬁltration through
0.20 mm ﬁlter. The (b-GP solution was added slowly to
the cooled camptothecin/chitosan dispersion under
aseptic conditions.
2.4. Invitro release study
Four hundred and eighty milligrams of ﬁne camptothecin powder, was dispersed in a 10 ml chitosan
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solution and intimately mixed by stirring (4.5% w/w
loading). Immediately after the addition of b-GP
solution, the mixture was quickly transferred to a mold
to form gels with dimensions 5 15 15 mm3. The mold
was pre-coated with a polyethylene glycol solution to
facilitate removal after gelation. After an incubation
time of l h 30 min at 37 C, the gels were removed from
the mold. In this way a matrix containing homogeneously distributed drug was obtained.
In vitro release was performed under inﬁnite sink
conditions using the molded gel immersed at 37 C in
500 ml of phosphate buffer pH = 7.4 containing 0.6%
Tween 20. The dissolution system was shaken at
100 rpm. Samples were removed periodically and the
medium was replenished. Released drug was measured
by an HPLC analytical method.
2.5. Cells and tumors
RIF-1 cells were obtained from Dr. Richard Hill
(Ontario Cancer Institute) and were passaged using
standard tissue culture techniques in RPMI 1640 media
supplemented with 10% fetal bovine serum and 1%
antibiotics (all supplied by Gibco BRL). Cells were
trypsinized, collected by centrifugation and resuspended
in media (4 106 cells/ml) before being injected (50 ml)
subcutaneously into the backs of previously shaved
C3H mice. Tumors appeared within 10 days and
reached a volume of 94–130 mm3 within 3 weeks.
Tumor volumes were calculated from measurements
taken at three orthogonal angles using the formula
(abcp/6).
2.6. Treatment
Treatments were begun when the tumors reached a
volume of approximately 100 mm3. Tumor-bearing
adult female mice (20 g) were separated into 4 experimental groups (n ¼ 627) for the different treatments.
One group was an untreated control. Two groups were
injected intratumorally with chitosan/GP or with
chitosan/GP loaded with camptothecin. 10 ml of chitosan/GP or chitosan/GP/CPT solutions were injected at
room temperature using a 26 G needle inserted in the
center of the tumor. After injection, the needle was held
in place for 3–4 s before being withdrawn to prevent the
hydrogel from leaking out of the injection site. The
amount of camptothecin incorporated in the hydrogel
was such that the total dose administered was 24 mg/kg.
In the last experimental group, the mice were injected
intra-peritoneally with 50 ml of camptothecin to give a
dose of 60 mg/kg. Camptothecin was dissolved for
injection a mixture of 8.3% Cremophor EL/8.3%
ethanol in 0.75% saline. Tumor measurements were
made daily, and the mice were sacriﬁced when the endpoint (4 initial tumor volume) was reached. All
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animal procedures were conducted according to the
guidelines of the McGill University Animal Care
Committee.
2.7. Measurement of camptothecin by HPLC
Quantitative analysis was performed on a Hewlett
Packard (Series 1100) chromatographic system equipped
with an Autosampler, a solvent module, a UV Detector,
and a System HP ChemStations system. The column
was a reverse-phase Lichrosphere RP18 (Chromatographic Specialities Inc.) column, (particle size 5 mm,
4 250 mm). The HPLC system was eluted isocratically
with methanol: water (63:37; v/v) at room temperature.
The ﬂow rate of the mobile phase was 1.0 ml/min and
samples were measured at a wavelength of 370 nm. A
standard curve was constructed by plotting peak area
against concentration. The assay was found to be highly
accurate and reproducible, with a coefﬁcient of determination = 0.9999.
3. Results
3.1. In vitro release
Chitosan/GP was loaded with camptothecin 4.5%
(w/w) and triplicate samples of polymer gels were
incubated in phosphate-buffered saline solutions containing 0.6% of Tween 20, pH 7.4, 37 C. At intervals,
the supernatant fractions were removed and the
medium replenished to maintain the sink conditions.
The amount of drug in the supernatant samples was
quantiﬁed by HPLC and the cumulative percentage of
the loaded drug released in the supernatant fractions
was plotted versus time. The amount of drug loaded
initially in the polymer was conﬁrmed by extraction of
the polymer with methanol to release the residual
camptothecin.
The cumulative release of camptothecin versus time of
incubation is shown in Fig. 1. Eighty percent of the drug
was released from the implant over 30 days in buffer
containing 0.6% of Tween 20. Approximately 13% was
released in the ﬁrst 72 h. The drug release from the
formulated chitosan/GP gel was nearly linear under
inﬁnite sink conditions, indicating almost zero-order
release kinetics in the ﬁrst four weeks after an initial
burst of less than 5% in the ﬁrst day. The drug
remaining in the chitosan/GP which had been immersed
in buffer for 4 weeks was extracted with methanol and
when this amount was combined with that released over
the preceding 4 weeks it appeared that approximately
80% of intact drug loaded in the gels had been
recovered.
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Fig. 1. In vitro release proﬁles of BST-gel loaded with camptothecin,
4.5% by weight. BST-gel/CPT immersed in phosphate buffer pH =
7.4 containing 0.6% of Tween 20.
Fig. 2. Delay of RIF-1 tumor growth after intratumoral (24 mg/kg)
and intraperitoneal (6 mg/kg) injections of camptothecin. O—O No
treatment, ’—’ Blank BST-gel, B—B Intraperitoneal injection
camptothecin, 6 mg/kg, m—m Intra-tumoral Implant BST-gel/camptothecin, 24 mg/kg.
Table 1
TGD following camptothecin treatment by intra-tumoral implant or
intra-peritoneal injection
Treatment
(TGD)7S.D.
No treatment
BST-gel
BST-gel/camptothecin (0.45 mg)
Camptothecin i.p 6 mg/kg
6.570.9
6.871.1
25.072.7
7.771.3
3.2. Tumor treatment studies
To evaluate its antitumor efﬁcacy camptothecin
formulated in chitosan/GP, was injected intratumorally
using a RIF-1 mouse tumor model. The RIF-1 tumor
has proven to be a useful model for preliminary
screening of various compounds for efﬁcacy because of
its reproducible growth, non-immunogenicity in the
syngeneic host and low frequency of spontaneous
metastases. It was only weakly responsive to camptothecin administered by intra-peritoneal injection (Table 1).
The effect of the camptothecin containing biodegradable polymer implants on tumor growth delay (TGD)
was examined. The results of these studies are shown in
Table 1 and Fig. 2. The implanted hydrogel containing
4.5% camptothecin by weight was found to be more
effective than systemically delivered camptothecin in
delaying tumor growth (TGDs of 25 and 8 days,
respectively). Tumors injected with blank chitosan/GP
showed no inhibition of growth and had a similar TGD
(7 days) as untreated tumors, conﬁrming that the
hydrogel alone has no effect on the growth of this
tumor. The greater effectiveness of the implant is
Fig. 3. Body weight of mice given camptothecin by different
administrative routes. The control and the BST-gel only group of
mice were sacriﬁced within 7 days as their tumors reached four times
the initial volume. O—O No treatment, ’—’ Blank BST-gel, m—m
Intra-tumoral implant BST-gel/camptothecin, 24 mg/kg, B—B Control mice no tumor.
presumably due to slow release of the drug in the tumor
and the exposure of tumor cells to toxic drug
concentrations for a prolonged period of time which
causes more cell death than does the short drug
exposure resulting from systemic administration.
Toxicity of camptothecin was evaluated in C3 H mice
with the RIF-1 tumor on the basis of weight loss. None
of the experimental groups showed any signiﬁcant effect
of treatment on body weight as is shown in Fig. 3. No
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M. Berrada et al. / Biomaterials 26 (2005) 2115–2120
topical irritation or any signs of mechanical stress were
observed in the case of solid gel implants.
4. Discussion
We selected camptothecin as a model drug for this
study, because its insolubility in water, makes it difﬁcult
to administer systemically by other means and because
of the potential applications of camptothecin and the
insoluble camptothecin analogues in chemotherapy.
Additionally, the pharmacologically important lactone
ring of camptothecin and its analogs is unstable in the
presence of human serum albumin which results in the
conversion of the active drug to the inactive carboxylate
form bound to albumin [19–21]. This imposes a severe
pharmacokinetic limitation on the systemic use of
camptothecin and related compounds. An approach to
overcoming this and other shortcomings of camptothecin and its analogs, especially their high systemic toxicity
is to load it into a delivery system such as a chitosanbased formulations which will protect the drug from
hydrolysis and control its release over a prolonged
period. Since the active drug is dispersed and not
solubilized there is no possibility of chemical reaction
between the active drug and the excipients.
There are three primary mechanisms for the loaded
drug to be released from hydrogels: swelling, diffusion
and degradation. Drug release from chitosan/GP gel
with initial water content of 84% (w/w) occurs through
the diffusion of water through the polymeric matrix and
dissolution of the soluble fraction of the drug. Water is
taken up by hydrogels immediately after being exposed
to an aqueous media, the rate of water uptake
depending on the hydrophilicity of the polymer. As
the gel swells the encapsulated drug is released by
diffusion through pores. In a study of the release of
model compounds from chitosan/GP gels [16] it was
found that release occurred largely by diffusion but
could be accelerated by weight loss of the gels. Weight
loss however occurred much more rapidly than drug was
released. This suggested that during the ﬁrst few hours
there is leaching of excess GP and of water which does
not contribute to the physical crosslinking of the gel.
The physical three-dimensional structure of the gel does
not change with time suggesting there is no substantial
erosion of the polymer matrix. The third mechanism,
which involves degradation of the polymer matrix,
would only occur under in vivo conditions as a result
of enzyme activity.
It is known that chitosans with block structures and
lower degrees of deacetylation (DDAo75%) are more
readily biodegraded due to the presence of blocks of
glucosamine moieties containing acetyl groups that
serve as a substrate for lysozyme [22,23]. In the present
study, we used chitosan (DDA 85%) that has been
2119
shown to degrade in vivo in about 6 months. No effect
of the degree of deacetylation of chitosan on the in vitro
release kinetics was observed but the in vivo release
kinetics would be expected to be different since in this
case the release kinetics is inﬂuenced by both the
biodegradation of chitosan and by diffusion.
Camptothecin delivered systemically resulted in a
TGD value of 8 days only, presumably due to the short
half-life of the drug and to the fact that the amount that
can be injected is limited by systemic toxicity. Giovanella et al. [24] have shown that after intramuscular
injection of camptothecin in Swiss nude mice (NIH high
fertility strain), camptothecin plasma concentrations
decline in a multi-exponential manner, with a mean
terminal elimination half-life of about 10 h. At this rate,
>99.99% of camptothecin is expected to be eliminated
from the systemic circulation within about 3.5 days.
In this study estimation of toxicity of chitosan/GP or
chitosan/GP/CPT was based on changes in body weight
following hydrogel implant. Several published studies
describe the effect of implant of chitosan/GP on the
histology of the surrounding tissue. The effect of
implant in normal tissue has been described by
Molinaro et al. [25] as a mild non-speciﬁc inﬂammatory
reaction. In the present study the hydrogel was
implanted into the tumor. A report of the effect of
chitosan/GP/paclitaxel implanted into the EMT-6
tumor described the histology of the implanted tumors
as showing some degree of necrosis interspersed between
viable tumor tissue with necrosis generally decreasing
away from the center of the tumor [26]. This pattern was
seen for both chitosan/GP implanted tumors and for
those implanted with chitosan/GP/paclitaxel. In the case
of the RIF-1 tumor chitosan/GP without drug appears
to have no tumoricidal effect so we would not expect to
observe the extent of necrosis seen in the EMT6 tumor
following implantation of chitosan/GP. Histological
changes following implant of chitosan/GP or chitosan/
GP/CPT will be investigated in a projected study.
Chitosan has been shown to activate macrophages for
tumoricidal activity in mice and guinea pigs [27]. Again,
since we found no difference in the tumor response
between the untreated mice and those injected intratumorally with chitosan/GP it seems likely that chitosan/
GP does not induce this type of tumoricidal activity
against the RIF-1 tumor.
The effectiveness of the polymer implant in delaying
tumor growth clearly demonstrates the importance of
this delivery system in maintaining an inhibitory level of
drug over a long period of time. The main advantages of
the biodegradable polymer implant such as chitosan/GP
used for the delivery of camptothecin to the mouse
tumor are the high intra-tumoral concentrations of drug
attainable, low systemic toxicity and the extended period
of time over which the drug can be released in the
tumor. The dose of camptothecin delivered using the
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M. Berrada et al. / Biomaterials 26 (2005) 2115–2120
implants was 24 mg/kg, which is 3 times the mean lethal
dose for C3H mice, for the implants the delayed release
of the drug and localization in the tumor prevents toxic
systemic levels being reached whereas a similar dose
delivered by bolus injection would be lethal.
[8]
[9]
[10]
5. Conclusion
Local delivery of chemotherapeutic agent by controlled-release polymers is a new strategy with the
potential to maximize the anti-tumor effect of a drug
and reduce systemic toxicity. In this study, we have
demonstrated the effectiveness of using the biodegradable
chitosan polymer to deliver high doses of camptothecin
locally to a mouse tumor model. Growth of tumors
treated in this fashion was retarded for signiﬁcantly
longer periods than were tumors treated with systemically
administered camptothecin. Camptothecin delivered by
intra-tumoral implant showed no toxicity in terms of
weight loss. The system formulated with camptothecin
was found to be stable and the release proﬁles of a
formulation with chitosan and b-GP showed almost zeroorder release kinetics in the ﬁrst four weeks after an initial
burst of less than 5% in the ﬁrst day.
These ﬁndings show chitosan/GP gel to be a safe,
effective, homogeneous, injectable and stable formulation
for delivery of camptothecin and this approach represents
an attractive technology platform for the delivery of
other clinically important hydrophobic drugs such as
taxol and tetracycline. The mechanism of gelation, which
does not involve covalent cross-linkers, organic solvent or
detergents, combined with a controllable residence time,
renders this injectable biomaterial uniquely compatible
with sensitive chemotherapeutic agents.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
References
[21]
[1] Herben VM, ten Bokkel HWW, Beijnen JH. Clinical pharmacokinetics of topotecan. [Review]. Clin Pharmacokinet 1996;31:85–102.
[2] Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin
induces protein-linked DNA breaks via mammalian DNA
topoisomerase I. J Biol Chem 1985;260:14873–88.
[3] Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu
Rev Biochem 1989;58:351–75.
[4] Gallo RC, Whang-Peng J, Adamson RH. Studies on the antitumor activity mechanism of action on cell cycle effects of
camptothecin. J Natl Cancer Inst 1971;46:789–91.
[5] van Hattum AH, Pinedo HM, Schluper HM, Erkelens CA, Tohgo
A, Boven E. The activity proﬁle of the hexacyclic camptothecin
derivative DX-8951f in experimental human colon cancer and
ovarian cancer. Biochem Pharmacol 2002;64:1267–77.
[6] Lerchen HG, Baumgarten J, von dem Bruch K, Lehmann TE,
Sperzel M, Kempka G, Fiebig HH. Design and optimization of
20-O-linked camptothecin glycoconjugates as anticancer agents. J
Med Chem 2001;44:4186–95.
[7] Knight V, Koshkina MV, Waldrep JC, Giovanella BC, Gilbert
BE. Anticancer effect of 9-nitrocamptothecin liposome aerosol on
[22]
[23]
[24]
[25]
[26]
[27]
human cancer xenografts in nude mice. Cancer Chemother
Pharmacol 1999;44:177–86.
Gottlieb JA, Luce JK. Treatment of malignant melanoma with
camptothecin (NSC-100880). Cancer Chemother Rep 1972;56:103–5.
Moertel CG, Schutt AJ, Reitemeier RJ, Hahn RG. Phase II study
of camptothecin (NSC 100880) in the treatment of advanced
gastro-intestinal cancer. Cancer Chemother Rep 1972;56:95–101.
Muggia FM, Creaven PJ, Hansen HH, Cohen MH, Selawry OS.
Phase I clinical trial of weekly and daily treatment with
camptothecin (100880): correlation with pre-clinical studies.
Cancer Chemother Rep 1972;56:515–21.
Weingart JD, Thompson RC, Tyler B, Colvin OM, Brem H.
Local delivery of the topoisomerase I inhibitor camptothecin
sodium prolongs survival in the intracranial 9L gliosarcoma
model. Int J Cancer 1995;62:605–9.
Storm PB, Moriarty JL, Tyler B, Burger PC, Weingart JD.
Polymer delivery of camptothecin against 9L gliosarcoma: release,
distribution and efﬁcacy. J Neuro- Oncol 2002;56:209–17.
LaVan DA, Lynn DM, Langer R. Moving smaller in drug
discovery and delivery. Nat Rev Drug Discovery 2002;1:77–84.
Jain RA. The manufacturing techniques of various drug loaded
biodegradable poly(lactide-co-glycolide) (PLGA) devices. [Review]. Biomaterials 2000;21:2475–90.
Chenite A, Buschmann M, Wang D, Chaput C, Kandani N.
Rheological characterization of thermogelling chitosan/glycerolphosphate solutions. Carbohydr Polym 2001;46:39–47.
Jarry C, Chaput C, Chenite A, Renaud MA, Buschmann M,
Leroux JC. Effects of steam sterilization on thermogelling
chitosan-based gels. J Biomed Mater Res 2001;58:127–35.
Chenite A, Chaput C, Wang D, Combes C, Buschmann MD,
Hoemann CD, Leroux JC, Atkinson BL, Binette F, Selmani A.
Novel injectable neutral solutions of chitosan form biodegradable
gels in situ. Biomaterials 2000;21:2155–61.
Lee JY, Nam SH, Im SY, Park YJ, Lee YM, Seol YJ, Chung CP,
Lee S J. Enhanced bone formation by controlled growth factor
delivery from chitosan-based biomaterials. J Controlled Release
2002;78:187–97.
Fassberg J, Stella VJ. A kinetic and mechanistic study of the
hydrolysis of camptothecin and some analogues. J Pharm Sci
1992;81:676–84.
Hertzberg RP, Caranfa MJ, Holden KG, Jakas DR, Gallagher G,
Mattern MR, Mong SM, Bartus JO, Johnson RK, Kingsbury
WD. Modiﬁcation of the hydroxy lactone ring of camptothecin:
inhibition of mammalian topoisomerase I and biological activity.
J Med Chem 1989;32:715–20.
Burke TG, Mi Z. The structural basis of camptothecin interactions with human serum albumin: impact on drug stability. J Med
Chem 1994;37:40–6.
Aiba S. Studies on chitosan: 4 Lysozymic hydrolysis of partially
N-acetylated chitosans. Int J Biol Macromol 1992;14:225–8.
Tomihata K, Ikada Y. In vitro and in vivo degradation of ﬁlms of
chitin and its deacetylated derivatives. Biomaterials 1997;18:567–75.
Giovanella BC, Stehlin JS, Hinz HR, Kozielski AJ, Harris NJ.
Preclinical evaluation of the anticancer activity and toxicity of 9nitro-20(S)-camptothecin (Rubitecan). Int J Oncol 2002;20:81–8.
Molinaro G, Leroux JC, Damas J, Adam A. Biocompatibility of
thermosensitive chitosan-based hydrogels: an in vivo experimental
approach to injectable biomaterials. Biomaterials 2002;23:2717–22.
Ruel-Gariepy E, Shive M, Bichara A, Berrada M, Le Garrec D,
Chenite A, Leroux JC. A thermosensitive chitosan-based hydrogel
for the local delivery of paclitaxel. Eur J Pharm Biopharm
2004;57:53–63.
Tokoro A, Tatewaki N, Suzuki K, Mikami T, Suzuki K, Suzuki
M. Growth-inhibitory effect of hexa-N-acetylchitohexaose and
chitohexaose against meth-A solid tumor. Chem Pharm Bull
1988;36:784–90.